Cells are continuously exposed to diverse stimuli ranging from soluble endocrine and paracrine factors, to signaling molecules on neighboring cells. It is of great importance that these extracellular signals are correctly interpreted by the cell, in order to achieve an appropriate developmental or proliferative response. Receptors of the tyrosine kinase family play pivotal roles in this process. By binding to specific peptide ligands they are able to integrate these external stimuli with internal signal transduction pathways, contributing in this fashion to the ability of the cell to respond correctly to its environment. In this review, we will concentrate on the role of ErbB receptors as normal signal transducers and their contribution to the process of malignant transformation during tumor development.

ErbB proteins belong to subclass I of the superfamily of receptor tyrosine kinases (RTKs). There are four members of the ErbB family: epidermal growth factor (EGF) receptor (also termed ErbB1/HER1), ErbB2/Neu/HER2, ErbB3/HER3 and ErbB4/HER4. We will refer to them, henceforth, as the ErbB receptors. All family members have in common an extracellular ligand‐binding domain, a single membrane‐spanning region and a cytoplasmic protein tyrosine kinase domain. A family of ligands, the EGF‐related peptide growth factors, bind the extracellular domain of ErbB receptors leading to the formation of both homo‐ and heterodimers. Dimerization consequently stimulates the intrinsic tyrosine kinase activity of the receptors and triggers autophosphorylation of specific tyrosine residues within the cytoplasmic domain. These phosphorylated residues serve as docking sites for signaling molecules involved in the regulation of intracellular signaling cascades. Ultimately, downstream effects on gene expression determine the biological response to receptor activation.

ErbB receptors are expressed in a variety of tissues of epithelial, mesenchymal and neuronal origin, where they play fundamental roles in development, proliferation and differentiation. Moreover, deregulated expression of ErbB receptors, in particular ErbB1 and ErbB2, has …

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The ErbB signaling network: receptor heterodimerization in development and cancer

Design of the Women's Health Initiative clinical trial and observational study

Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential. However, analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium.

Purification and unique properties of mammary epithelial stem cells

Generalized Linear Models (2nd ed.)

The epithelial components of the mammary gland are thought to arise from stem cells with a capacity for self-renewal and multilineage differentiation. Furthermore, these cells and/or their immediate progeny may be targets for transformation. We have used both in vitro cultivation and a xenograft mouse model to examine the role of hedgehog signaling and Bmi-1 in regulating self-renewal of normal and malignant human mammary stem cells. We show that hedgehog signaling components PTCH1, Gli1 , and Gli2 are highly expressed in normal human mammary stem/progenitor cells cultured as mammospheres and that these genes are down-regulated when cells are induced to differentiate. Activation of hedgehog signaling increases mammosphere-initiating cell number and mammosphere size, whereas inhibition of the pathway results in a reduction of these effects. These effects are mediated by the polycomb gene Bmi-1 . Overexpression of Gli2 in mammosphere-initiating cells results in the production of ductal hyperplasia, and modulation of Bmi-1 expression in mammosphere-initiating cells alters mammary development in a humanized nonobese diabetic-severe combined immunodeficient mouse model. Furthermore, we show that the hedgehog signaling pathway is activated in human breast “cancer stem cells” characterized as CD44 + CD24 −/low Lin − . These studies support a cancer stem cell model in which the hedgehog pathway and Bmi-1 play important roles in regulating self-renewal of normal and tumorigenic human mammary stem cells. (Cancer Res 2006; 66(12): 6063-71)

https://cancerres.aacrjournals.org/content/canres/66/12/6063.full.pdf

Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells.

In an attempt to determine the requirement of essential fatty acid for dimethylbenz(a)anthracene-induced mammary tumorigenesis, rats were fed diets containing different levels of linoleate: 0.5, 1.1, 1.7, 2.2, 3.5, 4.4, 8.5, or 11.5%. Each diet contained 20% of fat by weight, with varying amounts of coconut oil and corn oil added to achieve the desired levels of linoleate. Mammary tumorigenesis was very sensitive to linoleate intake and increased proportionately in the range of 0.5 to 4.4% of dietary linoleate. Regression analysis indicated that a breakpoint occurred at 4.4%, beyond which there was a very poor linear relationship, suggesting the possibility of a plateau. From the intersection of the regression lines in both the upper and lower ranges, the level of linoleate required to elicit the maximal tumorigenic response was estimated to be around 4%. The differences in tumor yield could not be correlated with changes in prostaglandin E concentration in the mammary fat pads of normal animals maintained on similar diets, suggesting that linoleate may act by some other mechanism to stimulate mammary tumorigenesis.

https://cancerres.aacrjournals.org/content/canres/45/5/1997.full.pdf

Requirement of Essential Fatty Acid for Mammary Tumorigenesis in the Rat

The effect of the prostaglandin synthetase inhibitor indomethacin on the dietary fat enhancement of 7,12-dimethylbenz(a) anthracene-induced mammary tumorigenesis has been examined in female Sprague-Dawley rats. Rats were fed either a normal-fat or high-fat diet (5 or 18% corn oil, respectively) with or without 0.004% indomethacin, starting 3 days after a single intragastric intubation of 5 mg 7,12-dimethylbenz(a)anthracene. Results of this experiment demonstrated that indomethacin completely blocked the stimulatory effect of fat on tumorigenesis, as measured by a decreased tumor incidence, a decreased number of tumors per group, a decreased tumor size, and an increased latency. No effect of indomethacin was observed in rats fed the normal-fat diet. These data suggest that at least part of the stimulatory effect of polyunsaturated fat on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis may be mediated through an increased synthesis of prostaglandins.

https://cancerres.aacrjournals.org/content/canres/43/8/3559.full.pdf

Effect of the prostaglandin synthetase inhibitor indomethacin on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in rats fed different levels of fat.

Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture.

There is increasing evidence that individual isomers of conjugated linoleic acid (CLA) may have unique biological or biochemical effects. A primary objective of this study was to determine whether there might be differences in the anticancer activity of 9,11-CLA and 10,12-CLA. This was achieved by evaluating the reduction in premalignant lesions and carcinomas in the mammary gland of rats that had been treated with a single dose of methylnitrosourea and given 0.5% of either highly purified CLA isomer in the diet. Our results showed that the anticancer efficacies of the two isomers were very similar. At 6 wk after carcinogen administration, the total number of premalignant lesions was reduced by 33-36%. At 24 wk, the total number of mammary carcinomas was reduced by 35-40%. The concentration of each CLA isomer and its respective metabolites was analyzed in the mammary fat pad. Tissue level of 10,12-CLA was much lower than that of 9,11-CLA. The pool of metabolites from each isomer was very similar between the two groups and represented only a small fraction of total conjugated diene fatty acids. Feeding of 9,11-CLA resulted in minimal changes in other unsaturated fatty acids. In contrast, feeding of 10,12-CLA produced a wider spectrum of perturbations. Small but significant increases in 16:1 and 16:2 were detected; these were accompanied by decreases in 20:2, 20:3, 20:4, 22:4, and 22:6. The above observation suggests that 10,12-CLA might be more potent than 9,11-CLA in interfering with elongation and desaturation of linoleic and linolenic acids. In summary, our study showed that, at the 0.5% dose level, the anticancer activity of 9,11-CLA and 10,12-CLA was very similar, even though accumulation of 10,12-CLA in the mammary tissue was considerably less than that of 9,11-CLA. These confounding changes of the other unsaturated fatty acids in contributing to the effect of 10,12-CLA need to be clarified.

Conjugated linoleic acid isomers and mammary cancer prevention.

Preface Section 1: In vivo model systems 1 Mouse models for mammary cancer D Medina 2 Methods for the induction of mammary carcinogenesis in the rat using either 7,12-dimethylbenz(alpha)anthracene or 1-methyl-1-nitrosourea HJ Thompson 3 A comparison of the salient features of mouse, rat and human mammary tumorigenesis D Medina, HJ Thompson 4 Xenograft models of human breast cancer cell lines and of the MCF10AT model of human preneoplastic, proliferative breast disease FR Miller, GH Heppner 5 Implantation and characterization of human breast carcinomas in SCID mice Y Xu, et al Section 2: Special techniques for in vivo studies 6 The cleared mammary fat pat and the transplantation of mammary gland morphological structues and cells LJT Young 7 Preparing mammary gland whole mounts from mice SB Rasmussen, et al 8 Slow-release pellets (Elvax) for localized in situ treatments of mouse and rat mammary tissue GB Silberstein, CW Daniel 9 Intramammary delivery of hormones, growth factors and cytokines BK Vonderhaar, E Ginsburg 10 Hormonal stimulation of the mouse mammary gland D Medina, F Kittrell Section 3: In vitro model systems 11 Collagen gel method for the primary culture of mouse mammary epithelium W Imagawa, et al 12 Chemical carcinogen induced transformation of primary cultures of mouse mammary epithelial cells grown inside collagen gels RC Guzman, et al 13 Establishment of mouse mammary cell lines D Medina, FKittrell 14 Whole organ culture of the mouse mammary gland E Ginsburg, BK Vonderhaar 15 Working with the mouse mammary end bud CW Daniel, GB Silberstein 16 Isolation and culture of normal rat mammary epithelial cells KM Darcy, et al 17 Characterization of normal human breast epithelial cell subpopulations isolated by fluorescence-activated cell sorting and their clonogenic growth in vitro J Stingl, et al 18 Isolation and culture of human breast cancer cells from primary tumors and metastases SP Ethier, et al Section 4: Molecular analysis and gene transfer techniques 19 mRNA in situ hybridization in the mammary gland S Weber-Hall, T Dale 20 Application of in situ PCR to studies of the mammary gland RC Hovey, BK Vonderhaar 21 Transfection of primary mammary epithelial cells by viral and non viral methods M Li, et al 22 Direct gene transfer into the mammary epithelium in situ using retroviral vectors TA Thompson, MN Gould 23 Intraductal injection into the mouse mammary gland D-A Nguyen, et al 24 Adenoviral and transgenic approaches for the conditional deletion of genes from mammary tissues K-U Wagner, et al 25 Transplantation and tissue recombination techniques to study mammary gland biology GR Cunha, et al 26 Rescue of mammary epithelium of early lethal phenotypes by embryonic mammary gland transplantation as exemplified with insulin receptor null mice GW Robinson, et al

Margot M. Ip

Papers

Requirement of Essential Fatty Acid for Mammary Tumorigenesis in the Rat

Effect of the prostaglandin synthetase inhibitor indomethacin on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in rats fed different levels of fat.

Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture.

Conjugated linoleic acid isomers and mammary cancer prevention.

Methods in Mammary Gland Biology and Breast Cancer Research